The invention relates to the fields of diagnosis and therapy of cancers. More particularly, the invention relates to a protein which mediates cell cycle arrest upon damage to cellular DNA.
It has long been known that DNA-damaging agents induce a cell cycle arrest, allowing time for repair and thus protecting the organism from the deleterious consequences of mutation. In mammalian cells, these arrests are often dependent on the functionality of the p53 gene product, a transcription factor which is translationally and post translationally activated following DNA damage (reviewed in Cox, Levine, and Morgan). Because p53 is mutated in a large fraction of cancers of diverse type, it is thought that the tumorigenic process may be intimately related to the disruption of p53-mediated control of the cell cycle. Accordingly, there has been much effort to define the molecular links between DNA damage, p53 expression, and cell cycle regulation.
Cells treated with ionizing radiation or other DNA-damaging agents arrest in both G1 and G2, with a consequent decrease in the fraction of cells in S phase. In colorectal cancer cells and many other epithelial cell types, 15-40% of the cells arrest in G1 and the remainder arrest in G2/M. The G1 block is in part mediated by p21WAF1/CIP1/SDI1, a cyclin dependent kinase inhibitor, which is transcriptionally controlled by p53. Several studies have suggested that the G2/M block following DNA damage is also p53-dependent. However, the basis for this G2/M block, though accounting for the predominant form of arrest induced by radiation in many cell types, is unknown.
Thus, there is a need in the art for elucidation of the pathway by which p53 exerts its cell cycle arresting effects. There is also a need in the art for new diagnostic and therapeutic tools for evaluating and ameliorating human cancers.
It is an object of the invention to provide DNA molecules useful for diagnosing and treating human tumors.
It is another object of the invention to provide proteins useful for treating human tumors and for raising diagnostically useful antibodies.
It is yet another object of the invention to provide methods of suppressing growth of tumor cells.
It is an object of the invention to provide a method for screening potential therapeutic agents for treating cancer.
It is another object of the invention to provide methods for diagnosing cancer.
It is yet another object of the invention to provide a reporter construct, useful for screening potential antineoplastic agents.
It is an additional object of the invention to provide an antisense construct for inhibiting expression of a tumor suppressor gene.
It is still another object of the invention to provide antisense oligonucleotides for inhibiting expression of a tumor suppressor gene.
It is yet another object of the invention to provide methods for promoting growth of cells in which a tumor suppressor gene""s expression is inhibited.
It is another object of the invention to provide a method for assessing susceptibility to cancers.
It is an object of the invention to provide a method for detecting the presence of wild-type p53 protein in a cell.
It is still another object of the invention to provide methods for identifying compounds which specifically bind to p53-specific DNA binding sequences which regulate "sgr" expression.
It is an object of the invention to provide methods for screening for anti-cancer drugs.
It is another object of the invention to provide cell lines useful for screening for anti-cancer drugs.
These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention an isolated and purified subchromosomal DNA molecule is provided. The molecule encodes 14-3-3"sgr" protein ("sgr") as shown in SEQ ID NO: 2. The sequence of cDNA encoding "sgr" is shown in SEQ ID NO: 1.
In another embodiment of the invention an isolated and purified 14-3-3"sgr" protein is provided. The protein has a sequence as shown in SEQ ID NO: 2.
In still another embodiment of the invention a method of suppressing growth of tumor cells is provided. The method comprises administration of a 14-3-3"sgr" protein having a sequence as shown in SEQ ID NO: 2 to said cells.
In an additional embodiment of the invention a method of suppressing growth of tumor cells is provided. The method comprises administration to said cells of a DNA molecule which causes said cells to express 14-3-3"sgr", said DNA molecule having a sequence as shown in SEQ ID NO: 1.
According to another embodiment of the invention a method for screening potential therapeutic agents for the ability to suppress the growth of tumor cells by activating the expression of 14-3-3"sgr" is provided. The method comprises incubation of a potential therapeutic agent with a cell which contains a 14-3-3"sgr" reporter construct, said reporter construct comprising a 14-3-3"sgr" transcription regulatory region covalently linked in a cis configuration to a gene encoding an assayable product. Further, the method comprises measurement of the production of the assayable product. A potential therapeutic agent is identified as useful if it increases the production by the cell of the assayable product.
In still another embodiment of the invention a method for diagnosing cancer is provided. The method comprises testing a tissue to determine if the tissue expresses less 14-3-3"sgr" than normal tissue.
In another embodiment of the invention a method for diagnosing cancer is provided. The method comprises testing a tissue to determine if DNA in said tissue contains a mutant 14-3-3"sgr" gene.
In still another embodiment of the invention a 14-3-3"sgr" reporter construct is provided. The reporter construct comprises a 14-3-3"sgr" transcription regulatory region covalently linked in a cis configuration to a gene encoding an assayable product.
In another embodiment of the invention an antisense 14-3-3"sgr" construct is provided. The construct comprises: a transcriptional promoter; a transcriptional terminator; and a DNA segment comprising one or more segments of the 14-3-3"sgr" gene, said gene segment located between said promoter and said terminator, said DNA segment being inverted with respect to said promoter and said terminator, whereby RNA produced by transcription of the DNA segment is complementary to a corresponding segment of 14-3-3"sgr" RNA produced by human cells.
According to another embodiment a method of identifying a chromosome is provided. The method comprises the steps of contacting one or more chromosomes with a polynucleotide probe which comprises at least 11 contiguous nucleotides of the sequence shown in SEQ ID NO: 1; and detecting chromosomes which specifically bind to the polynucleotide probe, wherein a chromosome which specifically binds to the probe is identified as containing at least a portion of human chromosome 1.
In another embodiment of the invention a 14-3-3"sgr" antisense oligonucleotide is provided. The oligonucleotide comprises at least ten nucleotides complementary to a sequence present in 14-3-3"sgr" mRNA.
In yet another embodiment of the invention a triplex oligonucleotide is provided. The oligonucleotide comprises at least ten nucleotides complementary to a sequence present in a 14-3-3"sgr" gene.
In still another embodiment of the invention a method is provided for promoting growth of cells. The method comprises: administering a 14-3-3"sgr" antisense or triplex-forming oligonucleotide comprising at least ten nucleotides complementary to 14-3-3"sgr" mRNA or 14-3-3"sgr" gene, respectively, to said cells to inhibit the expression of 14-3-3"sgr". In an alternative method an antisense 14-3-3"sgr" construct is administered to said cells to inhibit the expression of 14-3-3"sgr". The construct comprises:
a. a transcriptional promoter;
b. a transcriptional terminator;
c. a DNA segment comprising one or more segments of the 14-3-3"sgr" gene, said gene segment located between said promoter and said terminator, said DNA segment being inverted with respect to said promoter and said terminator, whereby RNA produced by transcription of the DNA segment is complementary to a corresponding segment of 14-3-3"sgr" RNA produced by human cells.
In still another embodiment of the invention a method is provided for assessing susceptibility to cancers. The method comprises testing a tissue selected from the group consisting of blood, chorionic villi, amniotic fluid, and a blastomere of a preimplantation embryo, to determine if DNA in said tissue contains a mutant 14-3-3"sgr" gene.
In one embodiment a method is provided for detecting the presence of wild-type p53 protein in a cell, comprising the steps of: providing a cell lysate from a tissue of a human; incubating a DNA fragment comprising BDS-2 with the cell lysate to bind the DNA fragment to wild-type p53 present in the cell lysate; immunoprecipitating p53 protein to form a precipitate; determining the amount of the DNA fragment comprising BDS-2 present in the precipitate.
In another embodiment of the invention a method is provided for detecting the presence of wild-type p53 protein in a cell, comprising the steps of: providing a cell lysate from a tissue of a human; incubating the cell lysate with a DNA fragment comprising BDS-2 to bind the DNA fragment to wild-type p53 present in the cell lysate; removing all components of the cell lysate not bound to the DNA fragment; determining the amount of p53 bound to the DNA fragment.
In yet another embodiment of the invention a method is provided for identifying compounds which specifically bind to BDS-2 comprising the steps of: providing a test compound; incubating a DNA fragment comprising BDS-2 immobilized on a solid support with the test compound, to bind the test compound to the DNA fragment; determining the amount of test compound which is bound to the DNA fragment.
In even another embodiment of the invention a method is provided for identifying compounds which specifically bind to BDS-2 comprising the steps of: providing a test compound; incubating a BDS-2-containing DNA fragment immobilized on a solid support with the test compound and wild-type p53 protein to bind the wild-type p53 protein to the DNA fragment; determining the amount of wild-type p53 protein which is bound to the DNA fragment, inhibition of binding of wild-type p53 protein by the test compound suggesting binding of the test compound to the p53-specific DNA binding sequences.
In one embodiment of the invention, a method for screening test compounds to identify those which are potential anti-tumor agents is provided. The method comprises the steps of: determining DNA content of 14-3-3"sgr" gene-defective human cells incubated in the presence and in the absence of a test compound, wherein a test compound which causes DNA accumulation in the 14-3-3"sgr" gene-defective cell is identified as a potential anti-tumor agent.
In another embodiment of the invention, a different method of screening for potential anti-tumor agents is provided. The method comprises the steps of: determining viability or apoptosis of 14-3-3"sgr" gene-defective human cells incubated in the presence and in the absence of a test compound; selecting a test compound which causes cell death or apoptosis in the 14-3-3"sgr" gene-defective cell.
In yet another embodiment of the invention a homozygous 14-3-3"sgr" gene-defective human cell line is provided.
In still another embodiment of the invention a pair of isogenic cell lines is provided. The first cell line is a homozygous 14-3-3"sgr" gene-defective human cell line and the second cell line is a homozygous 14-3-3"sgr" gene-normal human cell line.
According to another embodiment of the invention a method is provided for detecting p53 activity in a human tissue. The method comprises: contacting (a) a reporter construct comprising a 14-3-3"sgr" transcription regulatory region covalently linked in a cis configuration to a gene encoding an assayable protein product, with (b) a cell lysate of a tissue of a human, under conditions suitable to transcribe RNA from the reporter construct and to translate the RNA to form protein; measuring production of the assayable product; wherein a cell lysate which increases the formation of assayable product identifies the tissue from which it was made as having wild-type p53.
In yet another embodiment of the invention a method is provided for detecting p53 activity in a cell. The method comprises: transfecting a test cell with a reporter construct comprising a 14-3-3"sgr" transcription regulatory region covalently linked in a cis configuration to a gene encoding an assayable protein product; subjecting the test cell to a DNA damaging agent; measuring production of the assayable product in the test cell; comparing the production of the assayable product in the test cell subjected to a DNA damaging agent to production of the assayable product in a test cell which has not been subjected to the DNA damaging agent; wherein a test cell which produces more assayable product when subjected to the DNA damaging agent than the cell which has not been subjected to the DNA damaging agent is identified as having wild-type p53.
According to another aspect of the invention a method of detecting the presence of a wild-type p53 protein in a cell is provided. The method comprises the steps of: providing a histological section from a human; incubating the section with a detectably-labeled DNA fragment which comprises BDS-2 (SEQ ID NO: 5) to bind said DNA fragment to wild-type p53 present in the histological sample; removing unbound DNA fragment from the histological section; and determining the amount of DNA fragment which is bound to the histological sample.
In another aspect of the invention a method of identifying compounds which specifically bind to a p53-specific DNA binding sequence is provided. The method comprises the steps of: contacting a DNA fragment which comprises BDS-2 (SEQ ID NO: 5) with a test compound to bind the test compound to the DNA fragment; determining the amount of test compound which is bound to the DNA fragment.
Thus the subject invention provides the art with useful means for diagnosing and treating cancers in humans and other animals. Moreover, it opens new avenues for the design and screening of additional anti-neoplastic therapeutic agents which operate by means of a new mechanism as detailed below. Conversely, the subject invention provides a new approach for promoting the proliferation of cells when large numbers of such cells are desired.